Dendrimers have received great attention in recent years due to their possible use in applications as varied as catalysis on a nanoscale, chemical sensors, unimolecular micelles, imitation of the enzyme function, molecule encapsulation, molecular recognition, diagnostic agents and also as vehicles for carrying of genes and drugs. Excellent reviews which include all these applications are published in the bibliography.[1,31-38]
One of the areas wherein the dendrimers have been most studied is Gene Therapy (introduction of genetic material in a cell with therapeutic purposes). Until now, the most widely used vectors for carrying nucleic acids have been viral ones. However, the use of viral vectors has been associated to the appearance of some problems such as adverse immunological effects, lymphoproliferative effects related to the deregulation of oncogenes in the human genome[2], etc. To attempt to resolve these problems other types of non-viral vehicles have been developed such as cationic liposomes, polymers and also, as has been mentioned, dendrimers. Each of these cationic systems forms electrostatic complexes with the nucleic acids which are called, respectively, lipoplexes polyplexes or dendriplexes.
The use of liposomes as transfection agents was initially described in 1987.[3] The method most used for the distribution of genes has been encapsulation in cationic lipids, to the point that some of these derivatives such as Cytofectin™ or Lypofectin™ are commercially available. These derivatives, however, have also shown secondary effects such as inflammatory reaction of the lungs and problems such as lack of transfection in the presence of serum.[4]
With regard to conventional degradable polymers, their use as vehicles has the main drawback of their thermodynamic instability, which makes the active species have a very short in vivo half-life.[5]
The greatest advantage of the dendrimers on the moiety of non-viral vehicles lies in a uniform structure and the possibility of modifying in versatile manner the skeleton and surface thereof, which allows the precise characterization of the complex (nucleic acid/vector) and a systematic research of the transfection process. The first publication which described the use of dentritic molecules as transfection agents appeared in 1993 describing the use of dendrimers called PAMAM[6] (polyamidoamine), and since then a great quantity of studies have been carried out.[7,39,40]. The use of these dendrimers as vehicles is based on the fact that at physiological pH some of the terminal groups are protonated giving the PAMAM dendrimer a net positive charge, although some amino groups also remain unprotonated. Good transfection results have also been achieved with these dendrimers, especially with dendrimers of sixth and seventh generation, however, the efficacy of this process can be increased by two or three orders of magnitude when PAMAM dendrimers activated by heat treatment are used, as is the case of Superfect™ or Polyfect™.
Another class of potential transfection agents are dendrimers which contain phosphorous atoms,[8] synthesized by Majoral et al. until the twelfth generation In this case, the surface of the dendrimers has been functionalized with protonated or methylated tertiary amines and have been tested as transfection agents of the luciferase gene of 3T3 cells. The efficacy increases on increasing the dendrimer generation until achieving a constant value between generations three and five. Furthermore, we should highlight that these dendrimers have better transfection efficacy in the presence of serum.
Finally, other macromolecules have been such, such as poly(propylenimine) (PPI)[9] or poly(lysine) dendrimers[10,41] as systems for the transport of DNA or oligodeoxynucleotides (ODN). For example, dendrimers from low generations of PPI have shown a certain capacity for in vitro transfection with low toxicity, although it has not been possible to use higher generations due to the increase in their toxicity.
For their part, oligonucleotides (ODN) are researched in medical applications in different fields. Thus, for example, antisense ODNs are short synthetic sequences (15-30 bases in length) of DNA or analogous which are complementary (or antisense) to a target sequence (an RNA sequence or the DNA sequence complementary to that from which that RNA could be transcribed); designed to interfere with a biological event, such as the transcription, translation or the cut and splice phenomenon[11]. These molecules are designed to interact as complementary sequences of target mRNA, preventing translation to proteins, by the degradation of the mRNA by the activity of the RNase or interfering with the reading by the ribosome. This is called “antisense therapy”. Antisense ODNs have been used in multiple fields since 1978 (antitumour therapy and infectious disease above all), until today when, after a period of doubt, the antisense ODNs have recovered their role as a powerful tool in Molecular Biology, especially from the approval by the American FDA of Formivirsen[12], an antisense ODN indicated in ocular infection by CMV in the context of HIV infection. Another antisense ODN, GEM231, is postulated as a molecule with potential application against different neoplasias[13]. This approach is also being researched for its possible use in isotopic labelling of tumours using Positron Emission Tomography[14]. There is, furthermore, a type of antisense ODN which it is postulated can act at a level of cellular DNA: they are triple helix forming ODNs, some of them designed for their application within the field of HIV[15].
Another totally different field is that of the application of ODN rich in non-methylated CpG sequences as immunomodulators. These sequences lead the immune response to a Th1 profile, characterized by an increased secretion of Interferon, Tumour necrosis factor, interleukine-2, and other factors which increase the immune system's capacity to eliminate pathogens such as virus and bacteria. These ODN interact with receptors of the lymphocyte surface such as those of the Toll-like receptor family. They are being researched to boost immune response in immunodeficient patients and in the context of allergic diseases, characterized by a Th2 balance, with the aim of carrying this profile to Th1[16].
One of the main problems of therapy with ODN is that of achieving adequate levels to attain the therapeutic effect. It is necessary to administer large quantities of ODN to achieve the biological effect, since they have a large affinity to bind to plasma proteins, such as albumin[17]. Binding to plasma proteins and other cell surface proteins is also considered responsible for some of the toxic effects of ODN in vivo (activation of the cascade of the complement, haemolysis, thrombocytopaenia, etc.)[18]. It is therefore believed that the use of a vehicle that prevents said binding to proteins could be translated in the production of greater levels of active ODN, furthermore prolonging the half-life thereof and decreasing its toxicity.
The problem of the interaction with proteins and their binding to them is also present in many other substances used as drugs, since proteins in general (and plasma proteins in particular: albumin, glycoproteins, lipoproteins) show functional groups which are potentially capable of interacting with substances present in the medium, including administered drugs. This bond is a determining factor for the distribution of said drugs, provided that the bound fraction of the drug, as it does not have the capacity to be transferred, does not form part of the vascular-tissue balance (“reservoir”), is not metabolized, is not excreted and has no effect (unless it is determined by said bond).
Plasma protein binding (PPB) is by far the most important and determining factor of drugs distribution, since binding to tissue proteins is, generally, very reduced. This is due, among other things, to the fact that the plasma concentration of proteins is much greater than the interstitial concentration of the tissues, whose proteins, furthermore, have very little mobility and less capacity to bind substances, the latter property of which is particularly notable in the case of albumin, a predominant protein in plasma in normal conditions and whereto acid drugs are mainly bound (although some are also alkaline), whilst the acid glycoproteins bind those alkaline ones.
Alkaline drugsAcid Drugs (Albumin)(Albumin - I1 acid glycoprotein)AspirinChlordiazepoxideFurosemideDiazepamPenicillinLidocainePhenytoinQuinineTolbutamideAmitryptilineWarfarin
As the albumin structure from the standpoint of the drug bond is quite complex, two main binding points or locus can be defined:
POINT I (“of Warfarin”):POINT II (“of Diazepam”):ChlorothiazideBenzodiazepinesFurosemideIbuprofenNalidixic AcidCloxacillinSalicylic AcidSalicylic AcidTolbutamideTolbutamideIndomethacinIndomethacin
Therefore, the problem of binding to plasma proteins does not only affect ODNs, but also almost all commonly used drugs. Many of these problems could also be avoided by the use of a vehicle. It seems clear that the vehicle to develop for this purpose must have a series of characteristics, such as, being:                Non-toxic        Non-immunogenic (unless to be used in vaccination)        Biocompatible        Have functional groups suitable to permit chemical fixation.        Limited corporal accumulation        Maintain the activity of the drug/ODN until reaching the site of action        
Furthermore, it seems obvious that the vehicle to develop must release the ODN or the drug transported over time, so that this could carry out this action. In this aspect, the development of vehicles which permitted the controlled release of ODN or determined drugs would be very desirable, in order to achieve maintained levels of active substance in the organism and the production of the effect gradually. For all these reasons, the use of dendrimers seems a possibility which meets the desired requirements, as they can act as vehicles of the active substances which would protect them from degradation by plasma enzymes and from interactions with proteins to which they could bind, increasing their blood levels and permitting a higher and/or more prolonged activity. ODNs in particular, like many drugs of interest, are anionic molecules (with negative charge), for which reason use as vehicles thereof of dendrimers with groups which facilitate their interaction and, especially, of those which are of cationic nature at physiological pH is a very suitable option to guarantee the stability of the complex during its transport. Therefore, the invention develops novel dendrimers, specifically of carbosilane type, and provides their use, among others, as vehicles for carrying ODNs and other anionic molecules of interest in the blood and/or in other bodily fluids. This involves a new field since no study has been published to date concerning the use of dendrimers with carbosilane structure, soluble in water as vehicles, although a report has been published on the in vitro biocompatibility of carbosilane dendrimers constituted on ethylene poly(oxide).[21] Furthermore, only three synthetic studies of cationic carbosilane dendrimers have been published to date.[22-24], none of them coinciding with those provided by the invention.
Among the drugs for which the dendrimers can act as vehicles, an interesting group is constituted by the cytotoxic drugs designed for tumour cells[73,74]. When this objective is sought, the dendrimers can be directed at the tumour cells with folic acid, which is overexpressed in the tumour cells, for which reason these dendrimers would have preference for their uptake by said cells with respect to the normal cells. Recently, PAMAM dendrimers modified with folate on their surface have been used as vehicles of boto isotopes in neurone capture therapies in cancer[75]. Furthermore, PAMAM dendrimers conjugated with cisplatin act as macromolecular platin vehicle, an anti-tumour drug, which is released from the dendrimer-platin complex in controlled form, giving rise to a greater accumulation thereof in solid tumours, with less toxicity than the free cisplatin[76]. Another alternative for controlled release is to establish covalent bonds between the dendrimer and the drug by biodegradable bonds at physiological pH, as has been proven with dendrimers with primary amines on the surface and modified partially with 1-bromoacetyl-5-fluorouracil to form a labile amide bond which is hydrolized in vitro at physiological pH, releasing 5-fluorouracil, a powerful antitumour agent, in controlled manner.
Other substances of interest in whose transport dendrimers may be of use are those which become toxic after being irradiated, due to the in situ formation of small quantities of oxygen in singlet state, which has deleterious physiological effects[69]. Articles have been published on dendrimers carrying photosensitive drugs, for example, with 5-aminolevulinic acid in the periphery, supposing these dendrimer agents to be promising in the treatment of keratinocyte tumours[70]. As candidates for the treatment of solid tumours, dendrimers have been evaluated based on polyarylether dendrimers carrying protoporphyrin as photosensitizer[71].
An additional group of drugs for which dendrimers could suppose interesting vehicles is constituted by drugs such as non-steroidal anti-inflammatory drugs, which have secondary effects such as gastrointestinal alterations or nephrotoxicity which could be avoided as they are supplied by transdermal route, instead of through the classic oral or parenteral routes. The data which indicate the presence of dendrimers, bound to the drugs to administer, lead to skin alterations which increase its permeability[72], become good candidates to be used in the transdermal administration of drugs.
The multivalence of the dendrimer's surface functional groups means the great variety of molecules which they can transport even include dendrimers with different functionalities: these are the tectodendrimers, which are being studied due to their great potentiality in possible biomedical applications.
The potentiality of dendrimers for drug transport is not only based on using the possible interactions with an external multivalent dendrimeric surface, but on the fact that the dendrimer's structure can be used to house the molecules which one wants to transport. An example of use of dendrimeric structure cavities is the so-called “dendritic box”[68], wherein a PPI dendrimer is modified on the surface with phenylalanine groups, which protect the external frame making it denser. During the dendrimer growth process, molecules of different sizes are encapsulated in its interior. The dendrimer can carry a different number of molecules according to the size thereof. When the dendrimer is treated with formic acid, the outer frame is opened, allowing the release of the molecules housed therein.
Another group of molecules for which the dendrimers may suppose suitable vehicles are molecules of low molecular weight (such as peptides) against which it is desired to generate an immune response in a subject but which, due to their small size, are scarcely immunogenic or lead to a weak response after being injected in the individual which it is desired to treat. This problem can be resolved by increasing their molecular weight, either by polymerization of by coupling to a high molecular weight vehicle (traditionally to protein). Dendrimers which have a very defined structure and many functional groups capable of binding antigens in their periphery represent a good alternative for manufacturing vaccines which have very defined immunogens and which are highly reproducible. In this line, MAP (multiple antigenic peptide) dendrimers[57,58] have been developed, which are wedge-shaped constructions formed by successive generations of lysine residues. These dendrimers have a large number of primary amines which can be coupled to low molecular weight antigens, with the intention of increasing their immunogenicity, avoiding the need to use vehicle proteins. MAP structures which contain T and B cell stimulating Plasmodium falciparum peptides have been used to produce immune responses against this parasite[59]. Furthermore, it has been demonstrated that MAP structures are processed through the antigen-presenting cells in the same way as the antigens derivatives of intracellular structures (such as, for example, virus), giving rise to a potent immune response, including the production of cytotoxic T cells[60]. The dendrimers of the invention, which have at ends of their branches moieties which contain amino groups, may also be of use in vaccination, either because they are coupled to low molecular weight antigens making use of amines present in moieties of ends of their branches, or because said moieties which contain at least one amino group themselves constitute low molecular weight antigens such as those of peptide nature.
MAP structures have also been used to transport non-peptidic antigens such as carbohydrates, haptens, etc., in the context of vaccines. Carbohydrates in particular are a class of important molecules in biological recognition. Glycodendrimers, prepared by mannose-isothiocyanate, sialic acid or lactose binding to the terminal amines of PAMAM dendrimers or lysine dendrimers, have been used as antigens for vaccines[64,65]. In glycodendrimers with the T-associated beta Gal 1-3 alfaGalNAc disaccharide antigen its binding capacity to lectin has also been tested (binding protein to carbohydrates) specific for galactose[66], with the intention of using them to detect tumours which express T antigen receptors and to carry drugs thereto. The glycodendrimers can be used, furthermore, to increase affinity for lectins which are bound to the carbohydrate they have bound[67], which may be of interest for using those glycosilated dendrimers as microbial anti-adhesins, toxin antagonists, or as anti-inflammatory, antiviral and anti-cancer drugs, since the lectin-carbohydrate interactions of carbon have been described in numerous cases in the immune system (in the events that lead to cell activation), in viral and bacterial infections, in relation to cancer and the cell growth, etc. In short, glycosylated dendrimers may imitate the natural glycoconjugates and efficiently interact with the natural receptors of carbohydrates, giving rise to characteristic effects of interaction therewith.
In addition to the possibility of making use of its properties to use them as vehicles, another field related to nucleic acids to which much attention is being paid at present is of the manufacturing of microchips which contain ordered sets of DNA or RNA sequences. When these microchips are manufactured, dendrimers are arising as one of the alternative to coat glass surfaces and make use of their capacity of interaction with the nucleic acids to fix said molecules to the surface of the microchips[63]. The durability of the bond between sequences of nucleotides and dendrimers of the invention makes them suitable to use their capacity of fixing nucleic acids in order to serve as a base for the manufacturing of these DNA or RNA microchips.
Finally, these is also a need to find alternative methods to fight against different pathogens, interfering in their life cycle, a field in which dendrimers are showing themselves to be an interesting alternative. Some previously described dendrimers have shown themselves to be capable of inhibiting the infection caused by different viruses, interfering both with the entry of virus in the cells and in the later steps of viral replication. That is the case, for example, of Herpes Simplex, whose infection is inhibited in vitro by the effect of modified polylysine dendrimers[50,51]. Replication of HIV has also been achieved, both at the level of cell uptake and in the later steps, in this case by the use of covalently modified PAMAM dendrimers, which demonstrated that they were capable of interfering with the retrotranscriptase and integrase of the virus[19,52]. Making use of these properties, vaginal gels have been developed for the prevention of sexually transmitted diseases with dendrimer-based formulations, as is the case of VivaGel™ (Starpharma), whose active ingredient is a polylysine dendrimer functionalized with naphthalene disulfonate moieties which seems to be effective in the prevention of HIV thanks to the capacity of binding to glycoprotein gp120 of the virus surface. Although in the design of antiviral dendrimers there is preference for those which have groups on their surface which imitate those which are present on the cell surface and which, therefore, are capable of competing with the cells for binding to the virus, a dendrimer has also been designed with surface amide groups which function as respiratory syncytial virus inhibitor, thought to be due to the formation of hydrogen bridges between the peripheral groups of the dendrimer with the virus fusion protein, for which reason it is to be expected that dendrimers functionalized with other groups, capable of forming hydrogen bridges with viral proteins involved in the interaction of the cell surface, are also capable of interfering with different virus, inhibiting the infection caused by them.
In other cases, dendrimers have been used as antibacterial agents or to destructure the cell membranes of some fungi. When they are designed for this purpose, there is preference for dendrimers with cationic groups on their surface, such as amines or tetraalkylammonium groups, which facilitate adherence of dendrimers to the bacterial membrane, causing bacteria lysis. This is the case of poly(propylenimine) (PPI) dendrimers with tertiary alkylammonium groups on their surface, which have demonstrated extensive bacterial activity against both Gram positive and Gram negative bacteria[53,54]. These dendrimers have greater bactericide capacity than other hyperbranched polymers. The dendrimers of the invention, also functionalized with moieties which contain amino groups, represent an option to be also used to destructure cell membranes of bacteria or fungus.
It has also been communicated that dendrimers have properties which allow them to act as protein denaturants. Certain types of dendrimers act by decreasing the dielectric constant and the viscosity of the water and disordering its regular structure by the reorganization of the water molecules on the dendrimer surface. This leads to damaging hydrophonic interactions, which is very destabilizing for most tertiary protein structures, causing its denaturing: this is the so-called “chaotropic” effect which have denaturing agents such as urea or guanidine chloride. A very interesting field wherein it is intended to apply this denaturing capacity of proteins is the use to dissolve prionic proteins, such as PrPSc[20]. Prionic proteins are capable of adopting a pathogen structure-formation which cause mortal neuropathies called spongiform encephalopathies (Creutzfeldt-Jakob's disease, mad cow disease”, ovine scrapie, etc.). These proteins form aggregates which are located in the brains of the affected individuals and are only soluble in solvents which contain detergents as chaotropic agents (typically 6M guanidine chloride). However, these aggregates can be solubilized by cationic dendrimers such as those of PPI and PAMAM: those of greater generation with greater number of amines on the surface are the most effective. Therefore, the novel dendrimers of the invention, also functionalized with moieties which contain amino groups, provide novel compounds to be used, both to dissolve prionic aggregates and in the therapy of other diseases in whose development the formation of pathogenic protein aggregates also occurs, and, for example, the aggregates of amyloid protein which appear in Alzheimer's disease[55,56].
In short, dendrimers are synthetic polymers with good properties for their use in biological applications: they predictably respond in solution, they can be largely modified to carry multiple ligands with biological activity, they can cross biological barriers and are manufactured with few structural defects. Therefore, their application is being studied in different preventive and therapeutic strategies including their use for the carrying of different drugs, the transfection of oligonucleotidic or polynucleotidic molecules, the design of vaccines, the administration as antibacterial, antifungal, antiviral drugs or even the relief of the symptoms of diseases of different etiology in whose development the formation of protein aggregates such as those originated by prions or the deposits of amyloid protein characteristic of Alzheimer's disease, is involved. The dendrimers of the present invention involve interesting alternatives for these areas of Biomedicine.